Carboxymethylmannoglucans and derivatives thereof

ABSTRACT

A carboxymethylmannoglucan comprising tetrasaccharide units represented by the following general formula (I) and salt thereof. Further, the invention discloses a carboxymethylmannoglucan derivative and salt thereof produced by subjecting part or the whole of mannose of the tetrasaccharide units to ring opening and subjecting part or the whole of glucose which constitute the main chain but have no mannose as a branch ##STR1## wherein R 1  to R 12  each represent a hydrogen atom or a carboxymethyl group. 
     The compounds are useful as carrier for delaying the disappearance of a drug in the blood and for enhancing the organotropism of the drug for a carcinoma.

This is a divisional application of Ser. No. 08/397,560, filed Mar. 2,1995, now U.S. Pat. No. 5,567,690 which is a continuation of abandonedSer. No. 08/136,039, filed Oct. 14, 1993, which is a divisional ofabandoned Ser. No. 07/934,501, filed Oct. 21, 1992, which is a 371 ofPCT/JP92/00184, filed Feb. 21, 1992.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to novel carboxymethylmannoglucans andderivatives and salts thereof. More particularly, the present inventionis concerned with a carboxymethylmannoglucan and derivatives and saltsthereof for use as a carrier useful for delaying the disappearance of adrug in the blood and for enhancing the organotropism of the drug for acarcinoma.

2. Background Art

An attempt to use a water-soluble polymer as a carrier for a drug hashitherto been made especially in the field of a pharmaceuticalpreparation, and many related techniques for this purpose have beenproposed in the art. In many of these proposals, use is made ofcellulose derivatives such as carboxymethyl cellulose, hydroxypropylcellulose and hydroxypropyl methylcellulose, and the dispersion andsustained release of the drug are intended by virtue of physical andchemical properties of these substances per se. While in these attemptsthe drug is mixed homogeneously with the cellulose derivatives as acarrier, the drug is not chemically bonded to the carrier.

In the so-called "technique for organotropism" wherein a drug isdelivered by a necessary amount at a desired time to a target organ,when a water-soluble polymer is utilized as a carrier for a drug, thedrug and the carrier should be chemically bonded to each other ratherthan mere mixing. Examples of such attempts include bonding of mitomycinC to dextran (Hitoshi Sezaki, Yakugaku Zasshi, 109, 611-621 (1989)),bonding of mitomycin C to mannan (Report in the 49th General Meeting ofThe Japanese Cancer Association, (1990), page 425, theme No. 2155),bonding of bleomycin to mannan (Report in the 49th General Meeting ofThe Japanese Cancer Association, (1990), page 425, theme No. 2154), etc.The present status, however, is that no sufficient development is madeon the technique wherein a polysaccharide type water-soluble polymer isnewly synthesized and a drug is chemically bonded to this polymer todeliver the drug.

SUMMARY OF TEE INVENTION

Under the above circumstances, the present inventors have aimed at apolysaccharide polymer comprising a mannoglucan and attempted tocarboxymethylate the mannoglucan and, as a result, have found that theresultant substance is a novel polysaccharide-type, water-solublepolymer which is useful as a carrier for use in a technique wherein adrug is delivered by chemically bonding a drug to the carrier,particularly a technique for delaying the disappearance in the blood ofthe drug and for enhancing the migration of the drug to a carcinoma.

Specifically, an object of the present invention is to provide apolysaccharide-type, water-soluble polymer having a drug through achemical bond and capable of properly delivering a drug.

Another object of the present invention is to provide apolysaccharide-type, water-soluble polymer useful for delaying thedisappearance of a drug in the blood and enhancing the organotropism ofthe drug for a carcinoma.

According to a first aspect of the present invention, there is provideda carboxymethylmannoglucan comprising tetrasaccharide units representedby the general formula (I) or salt thereof. ##STR2## wherein R¹, R², R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² which may be the same ordifferent each represent a hydrogen atom or CH₂ COOH.

The second aspect of the present invention provides acarboxymethylmannoglucan derivative comprising tetrasaccharide unitsrepresented by the general formula (III) or salt thereof: ##STR3##wherein R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³ and R²⁴which may be the same or different each represent a hydrogen atom, CH₂COOH, CH₂ CONR*R¹ R*² wherein NR* ¹ R *² represents a residue formed byremoving one hydrogen atom from an amino group of a drug which has anamino group and is represented by the general formula HNR*¹ R*², CH₂COOR*³ wherein OR*³ represents a residue formed by removing a hydrogenatom from an alcoholic hydroxyl group of a drug which has an alcoholichydroxyl group and is represented by the general formula HOR*³, or CH₂COO.1/2 Pt(NH₃)₂ ! wherein Pt represents a divalent platinum,

with the proviso that at least one of R¹³ to R²⁴ in the moleculerepresents CH₂ CONR*R*², CH₂ COOR*³ or CH₂ COO.1/2 Pt(NH₃)₂ !.

Further, the third aspect of the present invention provides a oxidizedcarboxymethylmannoglucan or derivative thereof comprising unitsrepresented by the general formula (IV) and/or units represented by thefollowing general formula (V) or salt thereof: ##STR4## wherein R²⁵,R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰ which may be the same or different eachrepresents a hydrogen atom or CH₂ COOH;

W¹ and W² each represent ═O or ═N--R*⁴ wherein R represents a residueformed by removing two hydrogen atoms from an amino group of a drugwhich has an amino group and is represented by the general formula H₂N--R*⁴ ;

A¹ and A² which may be the same or different each represent a grouprepresented by the formula (VI), (VII), (VIII) or (IX);

A³ and A⁴ which may be the same or different each represent a grouprepresented by the following formula (VII), (VIII) or (IX),

with the proviso that when the molecule consists of units represented bythe general formula (IV) alone, not all the A¹ and A² in the molecule donot represent the formula (VI); ##STR5## wherein X^(i1), X^(i2), X^(i3),X^(i4), X^(i5), X^(i6), X^(i7), X^(i8) and X^(i9) which may be the sameor different each represent a hydrogen atom or CH₂ COOH; and

W^(i1), W^(i2), W^(i3), W^(i4), W^(i5) and W^(i6) which may be the sameor different each represent ═O or ═N--R*⁴ wherein ═N--R*⁴ represent aresidue formed by removing two hydrogen atoms from an amino group of adrug which has an amino group and is represented by the general formulaH₂ N--R*⁴,

with the proviso that each suffix "i" of X^(i1) to X^(i9) and W^(i1) toW^(i6) in the formulae (VI), (VII), (VIII) and (IX) represents aninteger of 1 to 4, and A¹, A², A³ and A⁴ are generally herein referredto as "A^(i) ".

The fourth aspect of the present invention provides a carboxymethylring-opening-mannoglucan and derivative thereof comprising unitsrepresented by the general formula (X) and/or units represented by thefollowing general formula (XI) or salt thereof: ##STR6## wherein R³⁰,R³¹, R³², R³³, R³⁴, R³⁵, R³⁶ and R³⁷ which may be the same or differentrepresents a hydrogen atom, CH₂ COOH, CH₂ CONR*¹ R*², CH₂ COOR*³ whereinNR*¹ R*² and OR*³ each have the same meaning as that defined in formula(III), or CH₂ COO.1/2 Pt(NH₃)₂ ! wherein Pt represents a divalentplatinum;

B¹ and B² which may be the same or different each represent a grouprepresented by the formula (XII), (XIII), (XIV) or (IX);

B³ and B⁴ which may be the same or different each represent a grouprepresented by the formula (XIII), (XIV) or (XV),

with the proviso that when the molecule consists of units represented bythe general formula (X) alone, not all the B¹ and B² represent a grouprepresented by the formula (XII); ##STR7## wherein Y^(j1), Y^(j2),Y^(j3), Y^(j4), Y^(j5), Y^(j6), Y^(j7), Y^(j8), Y^(j9), Y^(j10),Y^(j11), Y^(j12), Y^(j13), Y^(j14) and Y^(j15) which may be the same ordifferent each represent a hydrogen atom, CH₂ COOH, CH₂ CONR*¹ R*² orCH₂ COOR*³ wherein NR*¹ R*² and OR*³ each have the same meaning as thatdefined in formula (III), or CH₂ COO.1/2 Pt(NH₃)₂ ! wherein Ptrepresents a divalent platinum;

with the proviso that each suffix "j" of Y^(j1) to Y^(j15) in theformulae (XII), (XIII), (XIV) and (XV) represent an integer of 1 to 4,and B¹, B², B³ and B⁴ are generally herein referred to as "B^(j) ".

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a change in the concentration of a specimen inplasma when the specimen has been administered to rats bearing Walker256 at a dose of 18.0 μg/kg;

FIG. 2 is a graph showing a change in the concentration of a specimen inplasma when the specimen has been administered to rats bearing Walker256 at a dose of 10 mg/kg;

FIG. 3 is an ultraviolet-visible absorption spectrum of an oxidizedcarboxymethylmannoglucan-daunorubicin conjugate through a Schiff'sbase-type bond prepared in Experiment Example 2;

FIG. 4 is an ultraviolet-visible absorption spectrum of acarboxymethylmannoglucan-daunorubicin conjugate through an amide-bondprepared in Experiment Example 3;

FIG. 5 is an ultraviolet-visible absorption spectrum of acarboxymethylmannoglucan-mitomycin C conjugate through an amide groupprepared in Example 15;

FIG. 6 is an ultraviolet-visible absorption spectrum of a carboxymethylring-opening-mannoglucan-mitomycin C conjugate through an amide groupprepared in Example 18;

FIG. 7 is a gel filtration chromatogram of a carboxymethylring-opening-mannoglucan-mitomycin C conjugate through an amide groupprepared in Example 18;

FIG. 8 is an ultraviolet-visible absorption spectrum of an oxidizedcarboxymethylmannoglucan-daunorubicin conjugate through a Schiff'sbase-type bond prepared in Example 23;

FIG. 9 is an ultraviolet-visible absorption spectrum of an oxidizedcarboxymethylmannoglucan-daunorubicin conjugate through a Schiff'sbase-type bond prepared in Example 24;

FIG. 10 is an ultraviolet-visible absorption spectrum of an oxidizedcarboxymethylmannoglucan-daunorubicin conjugate through a Schiff'sbase-type bond prepared in Example 25; and

FIG. 11 is a gel filtration elution pattern of a cis-diammine-platinum(II) complex through a covalent bond prepared in Example 26.

DETAILED DESCRIPTION OF THE INVENTION

Compound

The carboxymethylmannoglucan according to the first aspect of thepresent invention comprises tetrasaccharide units represented by thegeneral formula (I). The term "comprises tetrasaccharide units" usedherein is intended to mean that the carboxymethylmannoglucan accordingto the present invention is a polymer compound having a structurecomprising said units as repeating units.

The tetrasaccharide units represented by the general formula (I) has abasic skeleton represented by the following formula (II). Hence, thecarboxymethylmannoglucan according to the present invention has a basicskeleton of a polysaccharide comprising tetrasaccharide unitsrepresented by the following formula (II). ##STR8##

This polysaccharide polymer has been already reported by one of thepresent inventors (Inoue K. et al., Carbohydrate Res., 114, 245-256,(1983)).

The polysaccharide having this basic skeleton is a D-manno-D-glucan, andthe main chain of the polysaccharide is a glucan having a β (1→4) bond.The polysaccharide has such a structure that an D-mannosyl group isbonded to every other D-glucose residue at its 3-position and 6-positionthrough an α (1→3) bond and an α (1→6) bond to form double branching.

The structure can be represented by the following formula besides theformula (II). ##STR9##

The molecular weight of the carboxymethylmannoglucan according to thepresent invention is not limited so far as the carboxymethylmannoglucancomprises tetrasaccharide units represented by the general formula (I).The molecular weight is preferably in the range from 1×10⁴ to 2×10⁶,still preferably about 1×10⁶.

The carboxymethylmannoglucan according to the present invention, inother words, has such a structure that the hydrogen atom of the hydroxylgroup in the above basic skeleton is substituted with a carboxymethylgroup. The proportion of introduction of the substituent can beexpressed by the degree of substitution defined in terms of the numberof substituents per saccharide residue. Specifically, it can beexpressed by the following equation: ##EQU1##

The upper limit of the degree of substitution is 3 in which all thehydroxyl groups are substituted. In the present invention, the degree ofsubstitution is preferably 0.01 or more. In the present invention, atleast one carboxymethyl group should be present in the molecule. In thissense, the compound having a degree of substitution of 0 is excluded. Itis needless to say that positions of introduction of the substituent inadjacent tetrasaccharide units may be the same or different so far asthe structure of individual tetrasaccharide units can be represented bythe general formula (I).

The carboxymethylmannoglucan according to the present invention canexist in the form of a salt. Suitable examples of the salt includealkali metal or alkaline earth metal salts, such as sodium salt,potassium salt and calcium salt, and amino acid salts such as argininesalt and lysine-salt.

The carboxymethylmannoglucan derivative according to the second aspectof the present invention is derived from a carboxymethylmannoglucanrepresented by the general formula (I). Specifically, the mannoglucanderivative comprising units represented by the general formula (III) hassuch a structure that in the general formula (I) a drug is carried onpart or the whole of the carboxymethyl group through an acid amide bond,an ester bond or a covalent bond.

Examples of the introducible drug include the following compounds.Specifically, drug which has an amino group and is represented by thegeneral formula HNR*¹ R*² are introducible through an acid amide bond,and specific examples thereof include daunorubicin, doxorubicin,mitomycin C and bleomycin. Drugs which have an alcoholic hydroxyl groupand are represented by the general formula HOR*³ are introduciblethrough an ester bond, and specific examples thereof includecyclocytidine, vincristine, vinblastine and adrenalin. Further, platinumcomplexes such as cisplatin are introducible through a covalent bond.

Although the molecular weight of the carboxymethylmannoglucan derivativeaccording to the second aspect of the present invention is not limited,it is preferably in the range from 1×10⁴ to 2×10⁶, still preferablyabout 1×10⁵. In any case, the proportion of introduction of thesubstituent, i.e., the degree of substitution is less than 3, and thelower limit exceeds zero (0). The degree of substitution is preferablyabout 1 to 2.

The carboxymethylmannoglucan derivative according to the second aspectof the present invention as well can exist in the form of a salt of thecarboxymethyl group. Examples of the favorable salt include alkali metalor alkaline earth metal salts, such as sodium salt, potassium salt andcalcium salt, and amino acid salts such as arginine salt and lysinesalt.

The oxidized carboxymethylmannoglucan and its derivative according tothe third aspect of the present invention comprise units represented bythe general formula (IV) and units represented by the general formula(V). The term "derivative" used herein is used only for a compoundwherein a drug has been introduced through a chemical bond. Therefore,the carboxymethylmannoglucan according to the third aspect of thepresent invention has a structure having an aldehyde group at itscleaved end formed by cleaving part or the whole of mannosyl groups oftetrasaccharide units constituting the carboxymethylmannoglucanrepresented by the general formula (I) and further cleaving part or thewhole of glucose residues which constitute the main chain but have nomannose as a branch. Further, it can be said that the oxidizedcarboxymethylmannoglucan derivative according to the third aspect of thepresent invention is such that a drug has been further introduced intothe aldehyde group through a Schiff's base-type bond.

In the general formula (IV), A¹ and A² each represent a grouprepresented by the formula (VI), (VII), (VIII) or (IX). In thisconnection, the case where the molecule consists of units represented bythe general formula (IV) alone and all the A¹ and A² are a grouprepresented by the formula (VI) is excluded. The group represented bythe formula (VII), (VIII) or (IX) is one formed by cleaving the bondbetween the 2-position and the 3-position of the mannose residuerepresented by the formula (VI), one formed by cleaving the bond betweenthe 3-position and the 4-position or one formed by cleaving the bondbetween the 2-position and the 3-position and the bond between the3-position and the 4-position.

In the general-formula (V), A³ and A⁴ each represent a group representedby the formula (VI), (VII), (VIII) or (IX).

In the formulae (VI), (VII), (VIII) and (IX), each suffix "i" of X^(i1)to X^(i9) and W^(i1) to W^(i6) in the formulae (VI), (VII), (VIII) and(IX) represents an integer of 1 to 4, and A¹, A², A³ and A⁴ aregenerally herein referred to as "A^(i) ". This means that, for example,a case where each of A¹ and A² branched from the same D-glucose is agroup represented by the formula (VII) and a case where two X^(i5),i.e., X¹⁵ and X²⁵, are independent of and different from each other fallwithin the scope of the present invention. Further, in units adjacent toeach other represented by the general formula (IV) and/or generalformula (V) in the molecule, R²⁵ to R³⁰ and A¹ to A⁴ and X^(i1) toX^(i9) and W^(i1) to W^(i6) may be different from each other.

Although the ratio of existence of a unit represented by the generalformula (IV) to a unit represented by the general formula (V), and theratio of existence of groups represented by the formulae (VI), (VII),(VIII) and (IX) are not particularly limited, they may be determined bytaking the kind and the hydrophilicity of the drug to be introduced intoconsideration.

Drugs which have an amino group and are represented by the generalformula H₂ N--R*⁴ are introducible through a Schiff's base-type bond,and specific examples thereof include daunorubicin, doxorubicin andbleomycin.

The carboxymethyl ring-opening-mannoglucan and derivative thereofaccording to the fourth aspect of the present invention comprises unitsrepresented by the general formula (X) and/or units represented by thegeneral formula (XI). The carboxymethyl ring-opening-mannoglucanaccording to the fourth aspect of the present invention has a structureformed by opening the ring of part or the whole of mannosyl groups oftetrasaccharide units constituting the mannoglucan and opening the ringof part or the whole of glucose residues which constitute the main chainbut have no mannose as a branch. Hereafter substituting with acarboxymethyl group part or the whole of the hydrogen atom of thehydroxymethyl group which is formed by the opening of the ring describedabove. Further, the carboxymethyl ring-opening-mannoglucan derivativeaccording to the fourth aspect of the present invention has such astructure that a drug is carried on the carboxymethyl group through anacid amide bond, an ester bond or a covalent bond.

In the general formula (X), B¹ and B² each represent a group representedby the formula (XII), (XIII),(XIV) or (XV). In this connection, thecompound which consists of units represented by the general formula (X)where B¹ and B² are a group represented by the formula (XII) is excludedfrom the present invention because the compound is D-manno-D-glucan perse. The groups represented by the formula (XIII), (XIV) or (XV) are oneformed by cleaving the bond between the 2-position and the 3-position ofthe mannose residue represented by the formula (XII), one formed bycleaving the bond between the 3-position and the 4-position or oneformed by cleaving the bond between the 2-position and the 3-positionand the bond between the 3-position and the 4-position.

In the general formula (X), B³ and B⁴ each represent a group representedby the formula (XIII), (XIV) or (XV). In this connection, a case whereB³ and B⁴ each represent a group represented by the formula (XII) isexcluded. This is because when the ring opening of the mannoglucan byoxidation is conducted, the mannose as a branched saccharidepreferentially undergoes cleaving through oxidation over D-glucose whichconstitute the main chain but has no mannose as a branch.

In the formulae (XII), (XIII), (XIV) and (XV), suffix "j" of Y^(j1) toY^(j15) represents an integer of 1 to 4, and B¹, B², B³ and B⁴ aregenerally herein referred to as "B^(j) ". This means that, for example,a case where each of B¹ and B² branched from the same D-glucose is agroup represented by the formula (XIII) and a case where two Y^(j5),i.e., Y¹⁵ and Y²⁵, are independent of and different from each other aswell fall within the scope of the present invention. Further, in unitsadjacent to each other represented by the general formula (X) and/orgeneral formula (XI) in the molecule, R³¹ to R³⁸ and B¹ to B⁴ and Y^(j1)to Y^(j15) may be different from each other.

Although the ratio of existence of a unit represented by the generalformula (X) to a unit represented by the general formula (XI), and theratio of existence of groups represented by the formulae (XII), (XIII),(XIV) and (XV) are not particularly limited, they may be determined bytaking the kind and the hydrophilicity of the drug to be introduced intoconsideration.

The carboxymethyl ring-opening-mannoglucan according to the fourthaspect of the present invention is favorable because it has a higherwater solubility than the carboxymethylmannoglucan according to thefirst aspect of the present invention while having the carboxymethylgroup into which a drug can be introduced.

Examples of the drug which can be introduced into the carboxymethylgroup of the part or the whole of the carboxymethylring-opening-mannoglucan according to the fourth aspect of the presentinvention through an acid amide bond, an ester bond or a covalent bondinclude drugs introducible into the mannoglucan derivative according tothe second aspect of the present invention.

Although the molecular weight of the carboxymethylmannoglucan and itsderivative according to the third aspect of the present invention andthe carboxymethyl ring-opening-mannoglucan and derivative thereofaccording to the fourth aspect of the present invention is not limited,it is preferably in the range from 1×10⁴ to 2×10⁶.

The proportion of introduction of the carboxymethyl group, i.e., thedegree of substitution is less than 3, and the lower limit exceeds zero(0). In the case of the oxidized carboxymethylmannoglucan and itsderivative, the degree of substitution is preferably 0.4 to 1.

The carboxymethylmannoglucan and its derivative and the carboxymethylring-opening-mannoglucan and derivative thereof as well can exist in theform of a salt of the carboxymethyl group. Favorable examples of thesalt include those as described with respect to thecarboxymethylmannoglucans according to the first and second aspects ofthe present invention.

Preparation of Compound and its Applications

The carboxymethylmannoglucan according to the first aspect of thepresent invention can be prepared by substituting a hydrogen atom of ahydroxyl group of a mannoglucan comprising tetrasaccharide unitsrepresented by the formula (II) with a carboxymethyl group.Specifically, it may preferably be prepared by reacting a mannoglucancomprising tetrasaccharide units represented by the formula (II) with ahalogenoacetic acid or its salt in the presence of an alkali. Forexample, it can be prepared by dissolving a starting compound in water,adding sodium hydroxide to the solution, adding monochloroacetic acidthereto while cooling, stirring the mixture at room temperature forabout 20 hr, adjusting the pH value to about 8 to 9, pouring thereaction mixture into methanol, collecting the resultant precipitate,washing the precipitate with methanol and acetone and drying the washedprecipitate. In this case, the degree of substitution of thecarboxymethyl group can be regulated through a variation in the amountof addition of the alkali and monochloroacetic acid or its salt.

The mannoglucan comprising tetrasaccharide units represented by theformula (II) may preferably be prepared from a purification productseparated from a filtrate of a culture of a microorganism belonging toActinomycete, for example, Microellobosporia grisea (Japanese PatentPublication No. 52402/1989).

The carboxymethylmannoglucan according to the first aspect of thepresent invention has a small rate of disappearance in the blood and anorganotropism prosperity for a carcinoma (for details, see the followingExperiment Examples). Since the carboxymethylmannoglucan according tothe present invention has many hydroxyl groups and carboxyl groups, adrug can be bonded through these functional groups. Therefore, thecarboxymethylmannoglucan according to the first aspect of the presentinvention can be used as a carrier useful in a technique wherein a drugis carried through a chemical bond for delivering the drug, particularlya technique wherein the rate of disappearance of drug in the blood islowered to enhance the migration of the drug to a carcinoma.

The introduction of the drug into the carboxymethylmannoglucan accordingto the present invention can be conducted through a selection of asuitable method depending upon the properties of the drug. For example,in the case of a drug having an amino group (for example, daunorubicinor doxorubicin), the introduction can be conducted by oxidizing thecarboxymethylmannoglucan according to the present invention withperiodic acid or the like to form an aldehyde group and bonding theretoa drug as a Schiff's base. Similarly, in the case of a drug having anamino group, it is also possible to bond the drug to the carboxyl groupthrough an amide bond. Furthermore, after the hydroxyl group isactivated with cyanogen bromide, a drug having an amino group is bondedthereto through an isourea bond. In the above methods, the drug havingan amino group may be one having in itself an amino group or one havingan amino group newly provided for the purpose of conducting bonding.Further, it is also possible to use a method which comprises selecting asuitable spacer having an amino group and adding the spacer to form acompound corresponding to a drug having an amino group.

Specific examples of the compound wherein a drug has been introducedinto the carboxymethylmannoglucan of the first aspect of the presentinvention include the carboxymethylmannoglucan derivative of the secondaspect of the present invention. The derivative to which a drug has beenintroduced through an acid amide bond or an ester bond may preferably beprepared by reacting a carboxymethylmannoglucan comprising unitsrepresented by the general formula (I) or its salt with a drugrepresented by the general formula HNR*¹ R*² or the general formulaHOR*³ under an acid amide bond or ester bond forming condition. Forexample, a derivative having daunorubicin introduced thereinto maypreferably be prepared by reacting carboxymethylmannoglucan withdaunorubicin hydrochloride in, for example, a borate buffer (pH: 8) inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) as a condensing agent and precipitating the productfrom ethanol. A derivative having a drug introduced through a covalentbond may preferably be prepared, for example, by reactingcis-dinitrate-diamine-platinum (II) as a platinum complex withcarboxymethylmannoglucan in an aqueous solution, conducting dialysis andprecipitating the product from ethanol.

The oxidized carboxymethylmannoglucan according to the third aspect ofthe present invention may preferably be prepared by subjectingcarboxymethylmannoglucan comprising units represented by the generalformula (I) to oxidation for ring opening. At first, an oxidizing agent(e.g., periodic acid or its salt) is added to thecarboxymethylmannoglucan with ice cooling, and a reaction is allowed tomoderately proceed at room temperature or below. The oxidizedcarboxymethylmannoglucan can be obtained, for example, by dialyzing thereaction mixture against water, adding sodium acetate as a precipitationagent, dropwise adding the mixture in ethanol to give a product as aprecipitate. It is also possible to convert the mannosyl group and theglucose residue to an aldehyde group to various degree through avariation in the amount of addition of periodic acid or its salt,thereby forming a carrier to which a desired amount of a drug can bebonded. Further, the degree of conversion to an aldehyde may beregulated through a variation in the reaction time, reaction temperatureand other parameters. With respect to the conversion to an aldehyde,reference may be made to Inoue K. et al., Carbohydrate Res., 123,305-314 (1983).

A drug represented by the general formula E₂ NR*⁴ can be reacted withthe carboxymethylmannoglucan thus prepared under a Schiff base-type bondforming condition to give a derivative having a drug carrier thereon.For example, a derivative having daunorubicin introduced as a drug maypreferably be prepared by reacting the oxidized carboxymethylmannoglucanwith daunorubicin hydrochloride in a borate buffer (pH: 8)/ethanol mixedsolution.

The carboxymethyl ring-opening-mannoglucan according to the fourthaspect of the present invention may preferably be prepared by openingthe ring of mannoglucan through oxidation, reducing an aldehyde groupformed by oxidation to form a hydroxymethyl group and introducing acarboxymethyl group into part or the whole of the hydroxymethyl group.For example, an oxidizing agent (e.g., sodium periodate) is added to anaqueous solution of mannoglucan, a reaction is moderately allowed toproceed at room temperature or below under a light shielding condition,and after the completion of the reaction, the reaction mixture isdialyzed against water. Then, sodium borohydride is added to the innersolution of dialysis, pH of the reaction mixture is adjusted to 5 andthen 7, the reaction mixture is dialyzed against water, and thenon-dialyzed solution is then concentrated to give a ring openedmannoglucan in a polyalcohol form. A carboxymethylring-opening-mannoglucan may preferably be prepared by dissolving thepolyalcohol in an aqueous sodium hydroxide solution, addingmonochloroacetic acid to the solution, allowing a reaction to proceed atroom temperature, adjusting the pH of the reaction mixture to 8 andpouring the reaction mixture into ethanol.

To the carboxymethyl ring-opening-mannoglucan thus obtained, a drug maypreferably be introduced as the same manner described with respect tothe carboxymethylmannoglucan derivative according to the second aspectof the present invention.

EXAMPLES Preparation

The mannoglucan as a starting compound in the following Examples wasproduced by Microellobosporia grisea (Institute for Fermentation, Osaka,deposit No.: IFO12518) as a production microorganism by the followingmethod.

The above strain was inoculated by means of a slant into a Sakaguchiflask containing 100 ml of a GC medium (2% glucose, 0.5% peptone, 0.5%corn steep liquor, 0.3% yeast extract, 0.5% sodium chloride, 0.3%calcium carbonate, 1.5% agar; pH 7.0), and cultivated at 28° C. for 5days while shaking. After 2 ml of the culture solution was inoculatedinto a Sakaguchi flask containing 100 ml of a GC medium, the cultivationwas similarly conducted for 3 days. 200 ml of the culture solution wasinoculated into a 30-liter jar fermentor containing 20 liters of aproduction medium (3% glucose, 2% corn steep liquor; pH 7.2), andstirred at 28° C. for 92 hr with aeration (10 liters/min, 250 rpm).Adekanol LG805 (manufactured by Asahi Denka Kogyo K.K.) was added as anantifoaming agent during the cultivation. The resultant medium washeated at 80° C. for 20 min, cooled to room temperature and filtered.The filtrate was passed through a column packed with Diaion PA306 (Cl⁻type). And then 0.5 liter of 10% cetyl→pyridinium chloride and 1.0 literof a 0.5M borate buffer (pH: 10) were added to the effluent andwashings. The resultant precipitate was collected, washed with water anddissolved in 2% acetic acid (2.0 liters). After ethanol (6.0 liters) wasadded to the solution, the resultant precipitate was collected. Theprecipitate was washed with ethanol and dissolved in a 0.02% aqueoussodium acetate solution (3.0 liters). The precipitation was againconducted from the centrifugal supernatant with ethanol. The collectedprecipitate was washed with 75%. ethanol, ethanol and acetone in thatorder and dried in vacuo over phosphorus pentaoxide at 50° C. for 8 hrto give 36 g of mannoglucan as an intended compound. The molecularweight (gel filtration method/standard substance: dextran, column:G5000PW) of the resultant mannoglucan was about 1×10⁶.

Example 1

Water (20 ml) and sodium hydroxide (1.05 g) were added to themannoglucan (500 mg) prepared in the Preparation 1 with stirring to givea clear solution. Monochloroacetic acid (1.5 g) was added and dissolvedin the solution with cooling, and a reaction was allowed to proceed atroom temperature for 20 hr with stirring. After the pH of the reactionmixture was adjusted to 8 with acetic acid, the mixture was poured intomethanol (80 ml). The resultant white precipitate was then collected byfiltration. The precipitate was washed with methanol and acetone in thatorder and dried in vacuo to give 481 mg of carboxymethylmannoglucan.This substance was designated as CM-1, and the degree of substitution(DS) per saccharide residue was measured according to the followingmethod and found to be 0.08.

Measurement of Degree of Substitution

The degree of substitution (DS) was determined on a free acid form bythe following back titration. Specifically, the carboxymethylmannoglucanprepared above was shaken together with 70% nitric acid/methanol (1:10V/V) at room temperature for 3 hr. washed with 80% methanol and methanolusing methyl red as an indicator, and dried to give a sample. Thissample was dissolved in a predetermined excess or a 0.1N aqueous sodiumhydroxide solution and subjected to back titration with 0.1Nhydrochloric acid using phenolphthalein as an indicator. DS wasdetermined by the following equation (I):

    DS=16.2 (A-B)/{ S-5.8 (A-B)!

wherein S (mg) is the amount of the sample, A (ml) is the predeterminedexcess of 0.1N sodium hydroxide and B (ml) is the value of backtitration of 0.1N hydrochloric acid.

Examples 2 to 4

The procedure of Example 1 was repeated, except that the amounts ofsodium hydroxide and monochloroacetic acid were varied as described inTable 1. The yield, degree of substitution and designated name of thesubstances prepared in Examples 1 to 4 are given in Table 1.

                  TABLE 1    ______________________________________         NaOH     MCA     Yield Degree of Designated    Ex.  (g)      (g)     (mg)  Substitution (DS)                                          Name    ______________________________________    1    1.05     1.5     481   0.08      CM-1    2    1.75     2.5     503   0.17      CM-2    3    2.45     3.5     524   0.31      CM-3    4    3.50     5.0     598   0.53      CM-4    ______________________________________     Note) MCA: monochloroacetic acid

Example 5

20 ml of water and 3.5 g of sodium hydroxide were added to 500 mg ofCM-4 (degree of substitution: 0.55) prepared in the same manner asExample 1 to give a clear solution. 5.0 g of monochloroacetic acid wasadded and dissolved in the solution with cooling, and a reaction wasallowed to proceed at room temperature for 20 hr. After the pH of thereaction mixture was adjusted to 8 with acetic acid, the mixture waspoured into 100 ml of methanol. The resultant precipitate was thencollected by filtration. The precipitate was washed with methanol anddried in vacuo to give CM-5 (531 mg). The degree of substitution of CM-5was 0.81.

Example 6

10 ml of water and 1.75 g of sodium hydroxide were added to CM-5 (250mg) prepared in Example 5 to give a clear solution. 2.5 g ofmonochloroacetic acid was added and dissolved in the solution withcooling, and a reaction was allowed to proceed at room temperature for21 hr. After the pH of the reaction mixture was adjusted to 8 withacetic acid, the mixture was poured into 60 ml of methanol. Theresultant precipitate was then collected by filtration. The precipitatewas washed with methanol and dried in vacuo to give CM-6 (261 mg). Thedegree of substitution of CM-6 was 1.0.

Experiment Example 1

(1) Sample and Specimen

CM-1 and CM-4 prepared in Examples 1 and 4 were used as a sample. Thefollowing experiment was conducted for preparing specimens for animaltest from each sample. Specifically, each sample was dissolved in water,0.5M sodium periodate was added in such an amount that the periodate ioncorresponds to 0.1 mol per mol of the saccharide residue of the sample.A reaction was allowed to proceed at room temperature for 25 hr, and thereaction mixture was dialyzed against water at 4° C. Sodium acetate wasadded to the inner solution, and the mixture was poured into a 4-foldvolume of ethanol. The resultant precipitate was washed with ethanol andacetone and dried. The powder thus obtained was reacted with sodiumborohydride labelled with tritium in a 2.5 mM aqueous sodium carbonatesolution at room temperature for 20 hr. After the pH of the reactionmixture was adjusted to 5 with acetic acid with cooling, the mixture wasdialyzed against water. The inner solution was lyophilized to givespecimen 1 and specimen 2.

(2) Method

i.) Maintenance of Tumor Cells

Walker 256 cells were intraperitoneally injected in an amount of 3×10⁶to 5×10⁶ cells to Wistar/S rats (6 to 9 weeks of age, ♀), and successionwas conducted every 7 days.

S-180 cells were intraperitoneally injected in an amount of 2×10⁶ to5×10⁶ cells to ICR mice (4 to 6 weeks of age, ♂), and passage wasconducted every 7 days.

ii.) Animals Bearing Tumors

Tumor cells were subcutaneously inoculated in an amount of 1.0×10⁷ cellsto the inguinal region of Wistar/S rats (6 weeks of age, ♀) , and therats were used as rats bearing Walker 256 6 days after the inoculation.

Tumor cells were subcutaneously implanted in an amount of 1.5×10⁶ cellsto the inquinal region of ICR mice (4 weeks of age, ♂), and the micewere used as mice bearing S-180 10 days after the implantation.

iii.) Distribution Study

Rats bearing Walker carcinosarcoma 256

Two experiments, i.e., one experiment under conditions of a dose of 18.0μg/kg and 6 hr and another experiment under conditions of a dose of 10mg/kg and 24 hr were conducted. A specimen was administered through thecervical vein of a rat bearing tumor under light ether anesthesia. Lightether anesthesia was conducted after a predetermined period of time,blood collection was conducted to determine the concentration of thespecimen in plasma. The rats were subjected to bloodletting to determinethe concentration of the specimen in the tumor and plasma 6 hr after theadministration in the case of a dose of 18.0 μg/kg and 24 hr after theadministration in the case of a dose of 10 mg/kg.

Mice bearing S-180

The specimen was administered at a dose of 18.0 μg/kg through a caudalvein of the mice bearing tumor. The mice were subjected to bloodlettingto determine the concentration of the specimen in the tumor and plasma 4hr after the administration.

The concentration of the specimen in the tumor and plasma weredetermined by burning the tumor and plasma in a combustion equipment andmeasuring the radioactivity by a liquid scintillation method.

(3) Results

The results are shown in FIG. 1, FIG. 2, Table 2 and Table 3. FIGS. 1and 2 are respectively graphs showing a change in the concentration of aspecimen in the plasma when the specimen was administered to the ratbearing Walker 256 at a dose of 18.0 μg/kg and at a dose of 10 mg/kg. Inthe Figures, a line of mark ▴ and a line of mark ◯ respectively show theresults of the specimen 1 and the specimen 2.

From the results shown in FIGS. 1 and 2, it is apparent that thesubstance according to the present invention does not rapidly disappearfrom the blood, and the diminution rate is low.

Tables 2 and 3 respectively show the concentration of the specimen inthe tumor and plasma, and the Kp value of the tumor tissue defined below(which is simply referred to as "Kp" in the table) for the specimen 1and the specimen 2. The Kp value is calculated according to thefollowing equation. ##EQU2##

From Tables 2 and 3, it is apparent that the substance of the presentinvention has the organotropism for a carcinoma.

                  TABLE 2    ______________________________________            Concentration                      Concentration            in Tumor  in Plasma            (ng/g)    (ng/ml)     Kp    ______________________________________    Mice bearing              7.60 ± 0.49                           2.36 ± 0.086                                      3.25 ± 0.30    S-180    (18.0 μg/kg,    4 hr)    Rats bearing              24.4 ± 3.83                          14.1 ± 1.72                                       1.73 ± 0.093    Walker 256    (18.0 μg/kg,    6 hr)    Rats bearing              19,210 ± 630                          575 ± 102                                      35.7 ± 6.31    Walker 256    (10 mg/kg,    24 hr)    ______________________________________

                  TABLE 3    ______________________________________           Concentration                     Concentration           in Tumor  in Plasma           (ng/g)    (ng/ml)     Kp    ______________________________________    Mice bearing             5.10 ± 0.43                         22.5 ± 1.16                                     0.226 ± 0.016    S-180    (18.0 μg/kg,    4 hr)    Rats bearing             26.7 ± 2.35                         97.5 ± 5.78                                     0.274 ± 0.017    Walker 256    (18.0 μg/kg,    6 hr)    Rats bearing             11,050 ± 619                         12,610 ± 1,110                                     0.891 ± 0.094    Walker 256    (10 mg/kg,    24 hr)    ______________________________________

Experiment Example 2 (Synthesis of Carboxymethylmannoglucan-DaunorubicinConjugate through Schiff Base-type Bond)

200 mg of CM-4 prepared in Example 4 was dissolved in 40 ml of water. Asolution of 21 mg of sodium periodate (corresponding to 0.1 mol per molof saccharide residue) dissolved in a small amount of water was addedthereto with stirring under ice cooling. A reaction was allowed toproceed at room temperature for 25 hr, and the reaction mixture wasdialyzed against water. 200 mg of sodium acetate was added to the innersolution, and the mixture was dropwise added to 350 ml of ethanol. Theresultant precipitate was collected, dried to give 192 mg ofcarboxymethylmannoglucan having aldehyde groups, and 20 mg of theproduct was dissolved in 4 ml of a 0.1M borate buffer (pH: 8.0) Asolution of 16.9 mg of daunorubicin hydrochloride dissolved in 4 ml ofethanol and 400 μl of a 0.1M borate buffer (pH: 8.0) was added thereto,and a reaction was allowed to proceed at room temperature overnight. 12ml of ethanol was added to the reaction mixture, and the resultantprecipitate was collected and dried to give 18 mg ofcarboxymethyl-mannoglucan-daunorubicin conjugate through a Schiff'sbase-type bond. This conjugate was soluble in water and had adaunorubicin content of 10.5% (% by weight). An ultraviolet-visibleabsorption spectrum (concentration: 200 μg/ml, solvent: water) of theconjugate. is shown in FIG. 3.

Experiment Example 3 (Synthesis of Carboxymethylmannoglucan-DaunorubicinConjugate through Amide Bond)

20 mg of CM-4 prepared in Example 4 was dissolved in 6 ml of a 0.1Mborate buffer (pH: 8.0). A solution of 5.6 mg of daunorubicinhydrochloride dissolved in 4 ml of ethanol and 1 ml of a 0.1M boratebuffer (pH: 8.0) was added thereto, and a solution of 60 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride dissolved in1 ml of water was further added. A reaction was allowed to proceed atroom temperature overnight. 24 ml of ethanol was added to the reactionmixture, and the resultant precipitate was collected and dried to give20 mg of a carboxymethylmannoglucan-daunorubicin conjugate wherein thedrug is bonded to the carboxyl group through an amide bond. Thisconjugate was soluble in water and had a daunorubicin content of 5.3% (%by weight). An ultraviolet-visible absorption spectrum (concentration:500 μg/ml, solvent: water) of the conjugate is shown in FIG. 4.

Example 7

Mannoglucan (3.00 g) was carboxymethylated in the same manner as that ofExample 4 to give 3.25 g of CM-4 (degree of substitution: 0.53). TheCM-4 (2.00 g) was carboxymethylated again in the same method as that ofExample 5 to give 2.22 g of CM-5 (degree of substitution: 0.79). TheCM-5 (1.00 g) was further carboxymethylated in the same manner as thatof Example 6 to give 1.08 g of CM-6 (degree of substitution: 1.0).

Example 8

CM-6 (500 mg) prepared in Example 7 was suspended in 2-propanol (30 ml),and the whole amount of a solution of 1 g of sodium hydroxide dissolvedin 3 ml of water was dropwise added thereto. Thereafter,monochloroacetic acid (1 g) was added, and a reaction was allowed toproceed at room temperature for 2 hr with stirring. The precipitate inthe reaction mixture was collected, and a reaction was again allowed toproceed at room temperature for 20 hr through the use of 2-propanol (40ml)/sodium hydroxide (1 g)-water (2 ml)/monochloroacetic acid (1 g). Theprecipitate in the reaction mixture was collected, dissolved in water(40 ml) and poured into methanol (240 ml), The resultant precipitate wascollected; washed with methanol and dried in vacuo to give 620 mg ofCM-7 (degree of substitution: 2.1).

Example 9

Mannoglucan (4.00 g) was dissolved in 0.1N hydrochloric acid (160 ml),the solution was subjected to acid degradation at 80° C. for 5 hr. andthe reaction mixture was neutralized with 5N sodium hydroxide. Thesolution was poured into ethanol (500 ml), and the resultant precipitatewas collected. The precipitate was washed with ethanol and dissolved inwater (250 ml). This solution was passed through both columns of Dowex50W-X2 (H⁺) and Dowex 1-X2 (Cl⁻) (each 1.5×20 cm), and the solutionpassed through the columns was concentrated to about 150 ml. Theconcentrate was poured into ethanol (500 ml), and the resultantprecipitate was collected and washed with ethanol and dried in vacuo togive 3.48 g of a low-molecular mannoglucan.

3.30 g of the low-molecular mannoglucan was dissolved in 1M sodiumchloride (330 ml), methanol (330 ml) was added thereto, and the mixturewas centrifuged. Methanol (110 ml) was added to the resultantsupernatant, and the resultant precipitate was collected. Theprecipitate was dissolved in water (50 ml) and poured into ethanol (200ml), and the resultant precipitate was collected, washed with ethanoland dried in vacuo to give 1.92 g of a low-molecular mannoglucan (MG15).The molecular weight of MG15 (gel filtration method/standard substance:dextran, column: G4000PW_(XL)) was about 1.5×10⁵.

Example 10

Mannoglucan (7.00 g) was dissolved in 0.1N hydrochloric acid (280 ml),the solution was subjected to acid degradation at 80° C. for 7.5 hr, andthe reaction mixture was neutralized with 5N sodium hydroxide. Thesolution was poured into ethanol (900 ml), and the resultant precipitatewas collected. The precipitate was washed with ethanol and dissolved inwater (450 ml). This solution was passed through both columns of Dowex50W-X2 (H⁺) and Dowex 1-X2 (Cl⁻) (each 2×20 cm), and the solution passedthrough the columns was concentrated to about 250 ml. The concentratewas poured into ethanol (850 ml), and the resultant precipitate wascollected and washed with ethanol and dried in vacuo to give 6.02 g of alow-molecular mannoglucan. 3.98 g of the low-molecular mannoglucan wasdissolved in 1M sodium chloride (400 ml), methanol (533 ml) was addedthereto, and the resultant precipitate was collected by centrifugation.The precipitate was dissolved in water (100 ml), and the solution waspoured into ethanol (400 ml). The resultant precipitate was collected,washed-with ethanol and dried in vacuo to give 2.00 g of a low-molecularmannoglucan (MG10). Further, methanol (267 ml) was added to thesupernatant obtained by the centrifugation just described above, and theresultant precipitate was collected. The precipitate was dissolved inwater (60 ml) and poured into ethanol (240 ml). The resultantprecipitate was collected, washed with ethanol and dried in vacuo togive 1.26 g of a low-molecular weight mannoglucan (MG4). The molecularweights of MG10 and MG4 (gel filtration method/standard substance:dextran, column: G4000PW_(XL)) were about 1×10⁵ and about 4×10⁴,respectively.

Example 11

MG15 (1.50 g) prepared in Example 9 was carboxymethylated in the samemanner as Example 4 to give 1.80 g of MG15-CM-4 (degree of substitution:0.52). 1.40 g of the MG15-CM-4 was carboxymethylated again in the samemethod as Example 5 to give 1.54 g of MG15-CM-5. The MG15-CM-5 (1.00 g)was further carboxymethylated in the same manner as Example 6 to give1.08 g of MG15-CM-6 (degree of substitution: 1.0).

Example 12

MG10 (1.80 g) prepared in Example 10 was carboxymethylated in the samemanner as Example 4 to give 2.23 g of MG10-CM-4. 2.00 g of the MG10-CM-4was carboxymethylated again in the same method as Example 5 to give 2.25g of MG10-CM-5. The MG10-CM-5 (1.0 g) was further carboxymethylated inthe same manner as Example 6 to give 1.07 g of MG10-CM-6 (degree ofsubstitution: 1.0).

Example 13

MG4 (500 mg) prepared in Example 10 was carboxymethylated in the samemanner as Example 4 to give 594 mg of MG4-CM-4 (degree of substitution:0.54).

Example 14

Mannoglucan (1.50 g) was dissolved in water (150 ml), a 8.5% aqueoussodium periodate solution (70 ml) was added thereto, and a reaction wasallowed to proceed at room temperature for 64 hr. Ethylene glycol (1.7g) was added to the reaction mixture, and the mixture was allowed tostand at room temperature for 2 hr and dialyzed against water. Sodiumborohydride (0.75 g) was added to the inner solution, and a reaction wasallowed to proceed at room temperature overnight. The pH of the reactionmixture was adjusted to 5 with acetic acid and then to 7 with 2N sodiumhydroxide. Thereafter, the mixture was dialyzed against water. The innersolution was concentrated to about 10 ml and poured into an ethanol (40ml)/acetone (80 ml) mixed solvent, and the resultant precipitate wascollected, washed with acetone and dried in vacuo to give 1.29 g of amannoglucan polyalcohol (MG-PA).

Water (1 ml) and sodium hydroxide (2.0 g) were added to MG-PA (500 mg)with cooling to give a clear solution. Monochloroacetic acid (2.9 g) wasadded and dissolved in the solution, and a reaction was allowed toproceed at room temperature for 18 hr. After the pH of the reactionmixture was adjusted to 8 with acetic acid, the mixture was poured intoethanol (200 ml). Then the resultant precipitate was collected. Theprecipitate was washed with water (5 ml) and poured into methanol (125ml). After that the resultant precipitate was washed with methanol anddried in vacuo to give 459 mg of a carboxymethylation product. 400 mg ofthe product was suspended in 2-propanol (40 ml), and the whole amount ofa solution of 0.8 g of sodium hydroxide dissolved in 1.6 ml of water wasdropwise added thereto. Thereafter, monochloroacetic acid (0.8 g) wasadded, and a reaction was allowed to proceed at room temperature for 20hr with stirring. The precipitate in the reaction mixture was collected,dissolved in water (8 ml) and poured into methanol (200 ml). Theresultant precipitate was collected, washed with methanol and dried invacuo to give 631 mg of a carboxymethylated mannoglucan polyalcohol(MG-PA-CM). The degree of substitution (DS) of MG-PA-CM was measured inthe same manner as Example 1 and found to be 9 per 4 saccharides. Inthis case, the DS value per 4 saccharides was determined by thefollowing equation.

    DS=59.4 (A-B)/ S-5.8 (A-B)!

Example 15

50 mg of CM-5 prepared in Example 7 and 50 mg of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) weredissolved in water (10 ml), and the solution was cooled with ice. Asolution of 4 mg of mitomycin C (MMC) dissolved in 0.8 ml ofwater-ethanol (1:1, v/v) was prepared, and the whole amount of thesolution was added to the above ice-cooled solution. A reaction wasallowed to proceed for 1 hr with ice cooling while maintaining the pH ofthe reaction mixture at 5 to 6 with 0.2N hydrochloric acid. After the pHof the reaction mixture was adjusted to 7.6 with 0.2N sodium hydroxide,the mixture was poured into ethanol (50 ml). Then the resultantprecipitate was collected, washed with 95% ethanol and dried in vacuo togive a conjugate comprising MMC bonded to CM-5 (52 mg). The MMC contentof the conjugate was 7.6% (% by weight) as determined with an absorptionat 365 nm. An ultraviolet-visible absorption spectrum of the conjugate(concentration: 96 μg/ml, solvent: water-ethanol (7:3, v/v) is shown inFIG. 5.

Example 16

CM-6 (50 mg) prepared in Example 7 was reacted with 4 mg of MMC by using50 mg of EDC in the same manner as Example 15 to give a conjugate (50mg) having a MMC content of 7.3% (% by weight).

Example 17

CM-7 (50 mg) prepared in Example 8 was reacted with 10 mg of MMC byusing 150 mg of EDC in the same manner as Example 15 to give a conjugate(57 mg) having a MMC content of 14% (% by weight).

Example 18

MG-PA-CM (30 mg) prepared in Example 14 was reacted with 10 mg of MMC byusing 150 mg of EDC in the same manner as Example 15 to give a conjugate(31 mg) having a MMC content of 24% (% by weight). Anultraviolet-visible absorption spectrum (concentration: 42 μg/ml,solvent: water-ethanol (7:3, v/v) and a gel filtration chromatogram ofthe conjugate were as shown in FIGS. 6 and 7, respectively.

Example 19

MG15-CM-4 (50 mg) prepared in Example 11 was reacted with 4 mg of MMC byusing 50 mg of EDC in the same manner as Example 15 to give a conjugate(53 mg) having a MMC content of 7.2% (% by weight).

Example 20

MG15-CM-6 (50 mg) prepared in Example 11 was reacted with 10 mg of MMCby using 150 mg of EDC in the same manner as Example 15 to give aconjugate (48 mg) having a MMC content of 15% (% by weight).

Example 21

MG10-CM-6 (50 mg) prepared in Example 12 was reacted with 10 mg of MMCby using 150 mg of EDC in the same manner as Example 15 to give aconjugate (55 mg) having a MMC content of 17% (% by weight).

Example 22

MG4-CM-4 (50 mg) prepared in Example 13 was reacted with 4 mg of MMC byusing 50 mg of EDC in the same manner as Example 15 to give a conjugate(46 mg) having a MMC content of 7.4% (% by weight).

Example 23

CM-4 (degree of substitution: 0.53, 1.25 g) prepared in the same manneras Example 4 was dissolved in water (300 ml). This solution was mixedwith a solution of 3.32 g sodium periodate (3 molar equivalents per moleof saccharide residue) dissolved in water (200 ml). After the reactionwas allowed to proceed at room temperature for one day, 1 g of ethyleneglycol was added to the reaction mixture. Then the further reaction wasallowed to proceed for 4 hr. The reaction mixture was dialyzed againstwater. And then the inner solution was concentrated. An ethanol-acetonemixed solution (about 1:1) was added to the concentrate, and methanolsaturated with sodium acetate (15 ml) was dropwise added thereto. Theresultant precipitate was collected to give 1.11 g of acarboxymethylmannoglucan having aldehyde groups. 800 mg of this productwas dissolved in a 0.1M borate buffer (pH=8.0, 250 ml), and the solutionwas mixed with 160 ml of an ethanol solution containing 130 mg ofdaunorubicin hydrochloride. The mixture was stirred at room temperaturefor 16 hr. After a 3M sodium chloride solution (8 ml) was added to themixture, the mixture was filtered. The filtrate was mixed with ethanol,and the resultant precipitate was collected to give 677 mg of acarboxymethylmannoglucan-daunorubicin composite. This conjugate wassoluble in water and had a daunorubicin content of 10% (% by weight). Anultraviolet-visible absorption spectrum (concentration: 330 μg/ml,solvent: water) was as shown in FIG. 8.

Example 24

CM-6 (800 mg) prepared in Example 7 was dissolved in water (200 ml). Thesolution was mixed with a solution of 2.12 g (3 molar equivalents permole of saccharide residue) of sodium periodate dissolved in water (30ml). After the reaction was allowed to proceed at room temperature forone day, 620 mg of ethylene glycol was added to the mixture. And then afurther reaction was allowed to proceed for 4 hr. The reaction mixturewas dialyzed against water, and the resulting inner solution wasconcentrated. An ethanol-acetone mixed solution (about 1:1) was addedthereto, methanol saturated with sodium acetate (10 ml) was dropwiseadded with stirring, and the resultant precipitate was collected to give643 mg of a carboxymethylmannoglucan subjected to conversion to analdehyde. 600 mg of the product was dissolved in a 0.1M borate buffer(pH=8.0, 175 ml), the solution was mixed with 110 ml of an ethanolsolution containing 150 mg of daunorubicin hydrochloride, and themixture was stirred at room temperature for 16 hr. A 3M sodium chloridesolution (4.5 ml) was added thereto, and the mixture was filtered. Thefiltrate was mixed with ethanol, and the resultant precipitate wascollected to give 610 mg of a carboxymethylmannoglucan-daunorubicinconjugate. The conjugate was soluble in water and had a daunorubicincontent of 13% (% by weight). An ultraviolet-visible spectrum(concentration: 200 μ/ml, solvent: water) of the conjugate is shown inFIG. 9.

Example 25

MG10-CM-6 (700 mg) prepared in Example 12 was dissolved in water (175ml). The solution was mixed with a solution of 1.86 g (3 molarequivalents per mole of saccharide residue) of sodium periodatedissolved in water (25 ml). After the reaction was allowed to proceed atroom temperature for one day, 560 mg of ethylene glycol was added to themixture. And then a further reaction was allowed to proceed for 4 hr.The reaction mixture was dialyzed against water, and the resulting innersolution was concentrated. An ethanol-acetone mixed solution (about 1:1)was added thereto, methanol saturated with sodium acetate (8 ml) wasadded thereto, and the resultant precipitate was collected to give 571 gof a carboxymethylmannoglucan subjected to conversion to an aldehyde.571 mg of the product was dissolved in a 0.1M borate buffer (pH=8.0, 180ml), the solution was mixed with 112 ml of an ethanol solutioncontaining 143 mg of daunorubicin hydrochloride, and the mixture wasstirred at room temperature for 16 hr. A 3M sodium chloride solution (3ml) was added thereto, and the mixture was filtered. The filtrate wasmixed with ethanol, and the resultant precipitate was collected to give610 mg of a carboxymethylmannoglucan-daunorubicin conjugate. Theconjugate was soluble in water and had a daunorubicin content of 12% (%by weight). An ultraviolet-visible spectrum (concentration: 200 μg/ml,solvent: water) of the conjugate is shown in FIG. 10.

Example 26

Cis-dinitratediammine-platinum (II) as a platinum complex wassynthesized by a known method (for example, Inorg. Chem., Vol. 16, p.1525 (1977), B. Lippert et al.).

MG-PA-CM (230 mg) prepared in Example 14 was dissolved in water (7 ml),a solution of cis-dinitratediammine-platinum (II) (24.71 mg) dissolvedin water (7 ml) was added thereto, and a reaction was allowed to proceedat room temperature for 24 hr under a light shielding condition. Afterit was confirmed by gel filtration chromatography that no platinumcomplex as a starting compound remained, the reaction mixture wasdialyzed against water overnight. After the pH of the inner solution wasadjusted to 6.5 with 1N NaOH, the solution was concentrated to about 10ml. Ethanol (80 ml) was added thereto, the resultant precipitate wascollected and dried in vacuo to give a cis-diammine-platinum (II)complex (218 mg, platinum content (by atomic absorption method): 5.90%).A gel filtration elution pattern (detection: ultraviolet absorption at280 nm) of the complex is shown in FIG. 11.

Example 27

A cis-diammine-platinum (II) complex (12.6 mg, platinum content: 7.04%)was prepared from CM-6 (14.6 mg) prepared in example 7 andcis-dinitratediammine-platinum (II) (2.12 mg) in the same manner asExample 26.

Example 28

A cis-diammine-platinum (II) complex (205 mg, platinum content: 6.60%)was prepared from CM-7 (211 mg) prepared in Example 8 andcis-dinitratediammine-platinum (II) (24.7 mg) in the same manner asExample 26.

We claim:
 1. An oxidized carboxymethylmannoglucan or derivative thereofcomprising units represented by the following general formula (IV)and/or units represented by the following general formula (V) or saltthereof: ##STR10## wherein R²⁵, R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰ which may bethe same or different each represents a hydrogen atom or CH₂ COOH;W¹ andW² each represent ═O or ═N--R*⁴ wherein R represents a residue formed byremoving two hydrogen atoms from an amino group of a drug which has anamino group and is represented by the general formula H₂ N--R*⁴ ; A¹ andA² which may be the same or different each represent a group representedby the following formula (VI), (VII), (VIII) or (IX); A³ and A⁴ whichmay be the same or different each represent a group represented by thefollowing formula (VI), (VII), (VIII) or (IX), with the proviso thatwhen the molecule consists of units represented by the general formula(IV) alone, not all the A¹ and A² in the molecule represent thefollowing formula (VI); ##STR11## wherein X^(i1), X^(i2), X^(i3),X^(i4), X^(i5), X^(i6), X^(i7), X^(i8) and X^(i9) which may be the sameor different each represent a hydrogen atom or CH₂ COOH; and W^(i1),W^(i2), W^(i3), W^(i4), W^(i5) and W^(i6) which may be the same ordifferent each represent ═O or ═N--R*⁴ wherein ═N--R*⁴ represent aresidue formed by removing two hydrogen atoms from an amino group of adrug which has an amino group and is represented by the general formulaH₂ N--R*⁴, with the proviso that each i of X^(i1) to X^(i9) and W^(i1)to W^(i6) in the formulae (VI), (VII), (VIII) and (IX) represents aninteger of 1 to
 4. 2. An oxidized carboxymethylmannoglucan or derivativeor salt thereof according to claim 1, which has a molecular weight of10,000 to 2,000,000.
 3. An oxidized carboxymethylmannoglucan orderivative or salt thereof according to claim 1 or 2, which has a degreeof substitution defined as the number of carboxymethyl groups persaccharide residue of 0.01 to 3.0.
 4. A process for preparing acarboxymethylmannoglucan or derivative or salt thereof according to anyone of claims 1 or 2, comprising the steps for reacting a mannoglucancomprising tetrasaccharide units represented by the formula (II)##STR12## wherein R¹⁰ represents hydrogen or CH₂ COOH, with ahalogenoacetic acid and reacting the resultant reaction product withperiodic acid or its salt.
 5. A process for preparing acarboxymethylmantioglucan or derivative or salt thereof according toclaim 3, comprising the steps of reacting a mannoglucan comprisingtetrasaccharide units represented by the formula (II) ##STR13## whereR¹⁰ represents hydrogen or CH₂ COOH, with a halogenacetic acid andreacting the resultant reaction product with periodic acid or its salt.6. A method for transferring a drug to a tumor in a mammal, comprisingthe step of administering an oxidized carboxymethylmannoglucan orderivative thereof comprising units represented by the following generalformula (IV) and/or units represented by the following general formula(V) or salt thereof: ##STR14## wherein R²⁵, R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰,which may be the same or different, each represents a hydrogen atom orCH₂ COOH;W¹ and W² each represent ═O or ═N--R*⁴, wherein R represents aresidue formed by removing two hydrogen atoms from an amino group of adrug which has an amino group and is represented by the general formulaH₂ N--R*⁴ ; A¹ and A², which may be the same or different, eachrepresent a group represented by the following formula (VI), (VII),(VIII) or (IX); A³ and A⁴ ₁, which may the same or different, eachrepresent a group represented by the following formula (VI), (VII),(VIII) or (IX), with the proviso that when the molecule consists ofunits represented by the general formula (IV) alone, not all the A¹ andA² in the molecule represent the following formula (VI); ##STR15##wherein X^(i1), X^(i2), X^(i3), X^(i4), X^(i5), X^(i6), X^(i7), X^(i8)and X^(i9), which may be the same or different, each represent ahydrogen atom or CH₂ COOH; and w^(i1), W^(i2), W¹³, W^(i4), W^(i5) andW^(i6), which may be the same or different, each represent ═O or═N--R*⁴, wherein ═N--R*⁴ represents a residue formed by removing twohydrogen atoms from an amino group of a drug which has an amino groupand is represented by the general formula H₂ N--R*⁴, with the provisothat each i of X^(i1) to X^(i9) and W^(i1) to W^(i6) in the formulae(VI), (VII), (VIII) and (IX) represents an integer of 1 to 4, ic to saidmammal.